HIV-1 infects the CNS early after primary infection. Although HAART has decreased the incidence of AIDS dementia, HIV-associated neurological disorders remain a problem. Only microglia and perivascular macrophages support productive viral infection in the brain, and these cell types express very low or undetectable levels of CD4. Sequence analysis has indicated that the virus may evolve independently in the CNS, probably resulting in adaptation to replication in the brain microenvironment. We have reported that a primary, peripheral HIV-1 isolate sequentially passaged in cultures of pure human adult microglial cells acquired an increased ability to replicate in microglia and induce syncytia formation, concurrently with higher ability to use low levels of CD4, increased exposure of CD4-induced epitopes, and increased sensitivity to neutralization. We hypothesize that viruses with envelopes conferring lower CD4/CCR5-dependence and higher neutralization sensitivity might arise in vivo due to the adaptation to replication in microglial cells. Using a panel of primary isolates obtained from CNS and peripheral autopsy tissues of HIV-l-positive individuals, we will investigate whether different conformations and significant changes in the interaction with cellular receptors, can be found between the envelopes from CNS and peripheral isolates. Thus, in Specific Aim 1 we will evaluate CD4 and co-receptor dependency, as well as sensitivity to neutralization and coreceptor and fusion inhibitors, in single-round pseudotype infections of cells with precisely regulated receptor expression levels. In Specific Aim 2 we will study the conformation of the envelopes, both in monomeric and oligomeric forms, with a panel of antibodies and several methodologies such as ELISA, FACS and surface plasmon resonance (SPR), which allows the analysis of interactions in real time. Finally, in Specific Aim 3 we propose to use SPR to study the interaction with CD4 and the chemokine receptors of soluble gp120 proteins derived from the primary CNS and peripheral isolates. These studies will provide insights into the in vivo evolution of HIV-1 and the mechanisms developed by the virus to increase the fitness in specific niches. Potentially, envelopes from primary viruses with partially-triggered conformation that are sensitive to neutralization, could be tested as immunogens with the goal of inducing a strong humoral immune response that might help to control HIV infection. [unreadable] [unreadable]